A low pKa ligand inhibits cancer-associated pain in mice by activating peripheral mu-opioid receptors

Abstract

The newly designed fentanyl derivative [( ±)-N-(3-fluoro-1-phenethylpiperidine-4-yl)-N-phenyl propionamide] (NFEPP) was recently shown to produce analgesia selectively via peripheral mu-opioid receptors (MOR) at acidic pH in rat inflamed tissues. Here, we examined the pH-dependency of NFEPP binding to brain MOR and its effects on bone cancer-induced pain in mice. The IC₅₀ of NFEPP to displace bound [³H]-DAMGO was significantly higher compared to fentanyl at pH 7.4, but no differences were observed at pH 5.5 or 6.5. Intravenous NFEPP (30-100 nmol/kg) or fentanyl (17-30 nmol/kg) inhibited heat hyperalgesia in mice inoculated with B16-F10 melanoma cells. The peripherally-restricted opioid receptor antagonist naloxone-methiodide reversed the effect of NFEPP (100 nmol/kg), but not of fentanyl (30 nmol/kg). The antihyperalgesic effect of NFEPP was abolished by a selective MOR- (cyprodime), but not delta- (naltrindole) or kappa- (nor-binaltorphimine) receptor antagonists. Ten-fold higher doses of NFEPP than fentanyl induced maximal antinociception in mice without tumors, which was reversed by the non-restricted antagonist naloxone, but not by naloxone-methiodide. NFEPP also reduced heat hyperalgesia produced by fibrosarcoma- (NCTC 2472) or prostate cancer-derived (RM1) cells. These data demonstrate the increased affinity of NFEPP for murine MOR at low pH, and its ability to inhibit bone cancer-induced hyperalgesia through peripheral MOR. In mice, central opioid receptors may be activated by ten-fold higher doses of NFEPP.

Figure 1

Competition of fentanyl and NFEPP with specific [³H]-DAMGO (4 nM) binding in mouse brain membranes at pH 7.4, 6.5 and 5.5. Concentration–response effects of (A) fentanyl and (B) NFEPP, where x represents the total specific binding in the absence of fentanyl or NFEPP. (C) IC₅₀ values of fentanyl (F) and NFEPP (NF). Means ± SEM (n = 6 in pH 7.4 groups, n = 5 per group for other pH values) are represented. **P < 0.01, compared to fentanyl at the same pH; ^●P < 0.05, ^●●P < 0.01 compared to NF at pH 7.4, two-way ANOVA and Tukey’s test.

Figure 2

Effects of i.v. fentanyl and NFEPP on heat hyperalgesia in mice inoculated with killed B16-F10 cells. (A) Dose–response effects of fentanyl (30–100 nmol/kg) and NFEPP (100–1000 nmol/kg) on ipsilateral paw withdrawal latencies (n = 5 per group). *P < 0.05, **P < 0.01 compared to solvent (0 nmol/kg)-treated mice, one-way ANOVA and Dunnett’s t test. (B) Systemic naloxone (NLX; 1 mg/kg s.c. at the neck) but not the local injection of naloxone-methiodide (NLX-MET; 1 µg s.c. in the calf) into the inoculated limb inhibited the antinociceptive effect of fentanyl (F) and NFEPP (NF) (n = 7 in F + NLX-MET group, n = 5 per group for other treatments). **P < 0.01 compared to the corresponding paw of the respective solvent (SOL)-treated mice, two-way ANOVA and Tukey’s test. All data are means ± SEM. For clarity, in the graphs a maximal number of two symbols of significancy are represented (the exact values are stated in the results section).

Figure 3

Effects of i.v. fentanyl and NFEPP on heat hyperalgesia in mice inoculated with live B16-F10 cells. (A) Dose–response effects of fentanyl and NFEPP (both at 10–100 nmol/kg) (n = 6 in NFEPP 30 nmol/kg group, n = 5 per group for other treatments). (B) Systemic naloxone (NLX; 1 mg/kg s.c. at the neck), but not peritumoral naloxone-methiodide (NLX-MET; 1 µg local), inhibited the effect of i.v. fentanyl (F; 30 nmol/kg). Both NLX (1 mg/kg) and NLX-MET (0.1–1 µg) reversed the effect of NFEPP (NF; 100 nmol/kg). NLX-MET (1 µg local) had no effect when injected into non-inoculated, contralateral limb (CL) (n = 6 in solvent [SOL] group, n = 8 in F + NLX-MET group, n = 5 per group for other treatments). (C) Peritumoral NLX-MET (1 µg local) did not modify antinociceptive effects evoked by high doses of fentanyl (F; 100 nmol/kg) or NFEPP (NF; 1000 nmol/kg) (n = 6 in SOL + NLX-MET and F + NLX-MET groups, n = 5 per group for other treatments). (D) Cyprodime (CYP; 1 mg/kg), but not naltrindole (NTI; 0.1 mg/kg) or nor-binaltorphimine (n-BNI; 10 mg/kg), injected s.c. at the neck, inhibited the effects of NF (100 nmol/kg) (n = 5 per group). In (A,B), ^●●P < 0.01 compared to the corresponding left paw, unpaired Student’s t test; **P < 0.01 compared to solvent (SOL)-treated mice, one-way ANOVA and Dunnett’s t test. In (C,D), ^●●P < 0.01 compared to the corresponding left paw, unpaired Student’s t test; **P < 0.01 compared to the corresponding paw in solvent (SOL)-treated mice, two-way ANOVA and Tukey’s test. All data are means ± SEM. For clarity, in the graphs a maximal number of two symbols of significancy are represented (the exact values are stated in the results section).

Figure 4

Effects of i.v. NFEPP (10–1000 mg/kg) on heat hyperalgesia in mice inoculated with NCTC 2472 or RM1 cells. Effects in mice inoculated with killed NCTC 2472 cells (n = 8 in 100 nmol/kg group, n = 7 in 300 nmol/kg group, n = 5 per group for other treatments) (A) or RM1 cells (n = 5 per group) (C), or in tumor-bearing mice inoculated with live NCTC 2472 cells (n = 6 in 30 nmol/kg group, n = 5 per group for other treatments) (B) or RM1 cells (n = 6 in 100 nmol/kg group, n = 7 per group for other treatments) (D). ⁺P < 0.05, ⁺⁺P < 0.01, compared to solvent (0 nmol/kg)-treated mice (A,C); *P < 0.05, **P < 0.01, compared to the solvent-treated mice in the right paw, one-way ANOVA and Dunnett’s t test (B,D). All data are means ± SEM. For clarity, in the graphs a maximal number of two symbols of significancy are represented (the exact values are stated in the results section).